A number of viruses are known to be of economic importance by reason of their role as the causative agent of certain diseases in animals. In the investigation of such viruses, and in development of vaccines and other materials for prophylaxis for such diseases, it has been a common practice to culture living animal cells in vitro. Many viruses, as well as various economically and scientifically significant microorganisms such as bacteria, rickettsiae and pleuropneumonia-like organisms (PPLO) have been made susceptible to laboratory examination by virtue of propagation in tissue culture of living animal cells. For example, many viruses have been isolated from diseased animals, propagated, and modified by attenuation or killed by specialized techniques after propagation by means including tissue culture as a central element. Tissue culture techniques are employed in the production of vaccines, in assay of viruses, in antibody assay, in interferon assay, in virus isolation procedures, in propagation of established virus strains, and a number of other techniques. Widespread use of tissue culture techniques has given economic significance to various cell lines, propagated in vitro for several successive transfers, which are employed in assay procedures, genetic studies, vaccine production and a number of other areas.
Many media for the propagation and maintenance of cells in tissue culture are known. Among these are a number of chemically-defined media, such as medium 199 of Morgan, Morton and Parker, Proc. Soc. Exptl. Biol. & Med. 73:1-8 (1950), Eagle's Basal Medium, Science 122:501-504 (1955), Science, 123:845-847 (1956), J. Biol. Chem. 226:191-206 (1957) and Eagle's Minimum Essential Medium, Science, 130:432-437 (1959) which may also include various balanced salt solutions (BSS) such as Hanks BSS, Earle BSS, Dulbecco Phosphate-buffered saline, Puck Saline F., Puck Saline G and the like. Merchant et al., Handbook of Cell and Organ Culture, Burgess Publishing Co., Minneapolis (1964). Such media materials consist of defined identifiable carbohydrates, minerals, amino acids, vitamins, salts, etc. in definite amounts, thus ensuring the identity of the medium from batch to batch. Other materials employed in tissue culture techniques, usually together with a chemically defined medium or balanced salt solution, include natural extracts and protein hydrolysates such as yeast extract, lactalbumin hydrolysate, Scherer maintenance medium, tryptone, tryptose, peptone, La Ye (lactalbumin hydrolysate-yeast extract) and the like. Such other extract and hydrolysate materials are not chemically defined, and are thus not susceptible of as wide application as are the synthetic chemically defined media. Both the chemically defined media and the natural extract and protein hydrolysate materials can be sterilized by autoclaving prior to use. A third class of material utilized in tissue culture techniques, often as a supplement to a chemically defined medium, are the natural animal protein materials such as albumins, globulins, or animal serum such as human, calf, bovine, lamb, rabbit serum or the like. The natural materials are characteristically neither chemically defined nor chemically pure. They can carry viral, rickettsial, PPLO, bacterial or other biological contaminants, and cannot be sterilized by heat without thermal degradation or inactivation of the desired animal protein. They may also be cytotoxic as respect some tissue, or they may carry undesired viral inhibitors or materials unsuitable for use in vaccine production or assay techniques.
Some of the problems inherent in the use of serum in virus production can be alleviated in many cases. For example, serum-containing growth media employed in growing cells for virus propagation can be replaced with a serum-free medium prior to inoculation with the virus to be propagated. However the replacement of the serum-containing medium typically requires repeated washing of the cells to ensure removal of the serum. The increased handling during the washings increases risks of contamination. Further, elaborate precautions must be taken to minimize the risk of contamination, thus making the multiple washing operation a tedious and expensive procedure. Propagation of the cells in an autoclavable serum-free or protein-free medium would eliminate the need for such washings, as well as elminating the risks and expense incurred in using serum in the growth medium. It would thus be desirable to provide a serum-free medium which can be used in both tissue culture growth and virus propagation.
Further, it is desirable to avoid the use of proteins and the like natural products since such materials are subject to considerable variability from batch to batch. Various autoclavable media for the growth of animal cells and for propagation of viruses have been employed. However, in most cases much more rapid and abundant propagation of cells and of viruses is obtained when such media are supplemented with animal serum or albumin. A chemically defined medium free of animal protein which provides enhanced cell and virus propagation would clearly be desirable.
A serum-free chemically defined medium comprising a buffered aqueous solution of carbohydrates, amino acids, mineral salts and vitamins, a water-soluble lipid source and a basic anion-exchange resin is described by Torney, U.S. patent application Ser. No. 725,517 filed Apr. 30, 1968. The medium is taught to be useful in growing Leptospira organisms.